In an infectious disease or autoimmune disease, an antibody against a specific antigen which is inherent in the disease appears in serum of a patient. In the conventional clinical test, it has simply been examined whether or not the serum contains the antibody. But in recent years, determination of the amount of the antibody and classification of its immunoglobulin class (IgG, IgA, IgM, IgD and IgE in case of human beings has attracted attention in order to investigate the cause of the disease or morbid state precisely.
For example, in case of an conventinal diagnosis of the infectious disease, the presence of the antibody against a pathogen of the infectious disease shows only the past history of the same infectious disease. However, if the change in an amount of the antibody and the class of immunoglobulin are ascertained, information concerning the time of infection or an advanced stage of the disease can be obtained. More specifically, as introduced in the journal of "Japan Clinics", Vol. 43, Autumn Special Issue, Second volume, page 27 (1985) with the title of "Interpretation of assay results of virus antibody", the detection of IgM antibody is required for the diagnosis of rubella.
Even if immunoglobulins to be assayed are in the same antibody class, it may be desirable to determine which kind of an antigen they react with among many kinds of antigens. For example, in the diagnosis of allergies the most important immunoglobulin class is IgE, and the RAST method (radioallergen adsorbent test) has been practiced as an available test method, wherein the kind of allergen as an etiogenic substance is identified and the amount of IgE reacting with the allergen is measured.
In this field RIA and EIA are both methods which have taken the leadership as a quantitative assay method. RIA and EIA are not only capable of carrying out a quantitative determination of high sensitivity, but also capable of identifying the immunoglobulin class to be assayed, so that they are significant in the diagnosis. However, these methods have disadvantages that they require to separate and wash the antibody reacted with the antigen so that the operation is complicated and takes much time. Also, in operations in which the sample should be treated many times, such as when separating and washing steps, are required, a danger of infection from the sample cannot be ignored. In addition, RIA involves many difficulties in handling, such as the problem in radioactive waste and the necessity for specific equipment. EIA has problems in that reaction time and temperature must be controlled strictly and that it is likely affected by an interfering reaction, since an enzyme is used as a labelling substance.
As to other assay techniques for the antibody in the field of the diagnostic, there can be mentioned passive blood cell agglomeration and a latex agglomeration, in addition to the above RIA and EIA. However, these methods have disadvantages in that they are limited only to a qualitative diagnosis, that the immunoglobulin class cannot be identified, and that the detection sensitivity is relatively low. For overcoming such problems partially, there is disclosed a method for detecting the immunoglobulin class of the antibody using an antigen-carrying magnetic particles and blood cells carying anti-globulin antibody (Japanese Patent Application Laid-Open (KOKAI) No. 177265/1985).
For identifying the class of the antibody to a specific antigen and determining the amount thereof present by using the above agglomeration method, it is necessary to measure the amount of agglomeration produced by the reaction between the antigen-carrying particles and the antigen-reactive antibody in the sample. In this event, the agglomeration of antibodies may sometimes occur due only to the antigen-carrying particle. In addition agglomeration of the particles carrying the anti-antibody which is specific to the immunoglobulin class of said antibody, may possibly occur as well due to immunoglobulins which are non-bindable to said antigen and are present in the sample serum in a large amount. Therefore, there still remain several problems without resolution. For example, undesirable steps which contain the most troublesome operations such as washing and resuspending are still required in the the subsequent steps. For example, the antigen-carrying particles is first reacted with a sample, and the reaction mixture is then separated and washed to remove the immunoglobulins which are non-bindable to said antigen, and then insoluble particles carrying the substance specific to the immunoglobulin class of the antibody are reacted therewith. Also, the time period for completing the assay is relatively long. Detection sensitivity is in addition inferior to that of RIA or EIA.
In the publication of WO 89/01161, there is disclosed a method in which magnetic material-containing particles carrying a monoclonal antibody thereon and an antigen in a specimen are reacted, followed by reaction with insoluble particles carrying an antibody thereon but containing no magnetic particles, then applying a magnetic field thereto to separate unreacted magnetic material-containing particles from agglomerated bulk comprising said two kinds of particles and the antigen, and measuring an amount of the remaining antibody-carrying insoluble particles. In this method the amount of the antigen in the specimen can be determined from the degree of decrease in turbidity. However, there is no description concerning the identification of the immunoglobulin class of the antibody. Thus, it has been desired to develop an assay method with high sensitivity, which can determine an amount of the antibody, can identify the immunoglobulin class of the same and can complete the assay within a short time.